Cellulase preparations containing reducing agent and method of processing fiber

ABSTRACT

A cellulase preparation comprising an endoglucanase derived from Zygomycetes, a cellulose-binding-domain-deleted endoglucanase, or a modified or homologous protein thereof, together with a reducing agent is disclosed. Further, a method of treating cellulose-containing fabric, comprising the step of treating the fabric with the cellulase preparation to improve a property of the fabric, a method of deinking waste paper, comprising the step of treating the waste paper with the cellulase preparation. together with a deinking agent, and a method of improving freeness of paper pulp, comprising the step of treating the paper pulp with the cellulase preparation, are disclosed.

TECHNICAL FIELD

The present invention relates to a cellulase preparation containing a reducing agent by which an endoglucanase activity is improved, and a method of treating a fabric using the cellulose preparation.

1. Background Art

Cellulase has three types of enzyme activities: a cellobiohydrolase activity which hydrolyzes solid crystal regions of cellulose from the nonreduced end in the exo manner to generate cellobiose; an endoglucanase activity which hydrolyzes amorphous regions of cellulose in the endo manner to transform cellulose molecules into low molecular weight molecules and to generate various types of cellooligosaccharides; and a β-glucosidase activity which decomposes cellobiose or cellooligosaccharides into glucose. Of these enzyme activities, it is known that cellulase having a high endoglucanase activity is advantageous when treating a fabric.

To impart desired properties to cellulose-containing fabric, the fabric has conventionally been treated with cellulase. For example, in the textile industry, treatment with cellulase is carried out to improve the touch and appearance of the cellulose-containing fabric, or to impart a “stonewash” appearance to the colored cellulose-containing fabric, thereby providing the fabric with localized color variations (EP Patent No. 307,564).

In such textile processing, cellulase derived from wood-rotting fungi such as Trichoderma or Humicola is mainly used. Such cellulase was used as a mixture comprising multiple cellulase components obtained by processing a culture filtrate of microorganisms having a cellulolytic activity. However, in order to achieve a greater economy, among cellulase preparations obtained by isolation from cellulase components, only endoglucanase, which mainly acts in fabric treatment, and which is genetically enhanced, has recently been used.

Examples of such an endoglucanase with a high activity include: EGV [Unexamined International Publication (Kohyo) No. 5-509223] and NCE4 (WO98/03640) derived from Humicola insolens, which strongly act on cotton fabrics; RCE I, RCE II, and RCE III derived from Rhizopus oryzae, which strongly act on lyocell fabrics; MCE I and MCE II derived from Mucor circinelloides; and PCE I derived from Phycomyces nitens (WO00/24879).

To improve the effects of the cellulase, the combined use of additives has also been attempted. For example, Unexamined International Publication (Kohyo) No. 5-507615 describes that a water-soluble polymer such as polyvinylpyrrolidone, polyvinyl alcohol, and polyacrylamide enhances the effects of Humicola insolens-derived cellulase and improves its activity of removing fuzz from colored fabrics. Further, it is known that a CMCase activity in the culture solution of Trichoderma viride is improved by the addition of Tween 20 (Ooshima, H. et al., Biotechnology and Bioengineering 28: 1727-1734, 1986). Furthermore, it is shown that the fuzz-removing activity of RCE I derived from Rhizopus oryzae is improved in the presence of a nonionic surfactant (WO02/38754).

DISCLOSURE OF THE INVENTION

The cellulases used for the above-described purposes are all expensive. Therefore, to achieve an industrial level application, the present inventors considered that a further improvement of the endoglucanase activity is desired, so that the above effects of cellulase can be more efficiently exerted. Further, where an attempt to obtain the effects of an improvement of the endoglucanase activity is carried out, the use of expensive additives increases costs in the textile processing treatment. Therefore, when selecting the additives, the present inventors considered that it is necessary to show the effects of the activity improvement by adding a low concentration of the additives, and that the additives should be readily available and inexpensive.

Therefore, the object of the present invention is to provide a cellulase preparation having the improved endoglucanase activity, which can be used in the fabric treatment for the purpose of improving cellulose-containing fabrics such that the removal of fuzz can be carried out efficiently and economically.

The present inventors have conducted intensive studies and, as a result, found that a reducing agent such as sodium thiosulfate or the like enhances the effects of Zygomycetes-derived endoglucanases such as RCE I, MCE I, PCE I, and the like at rates far higher than Trichoderma- and Humicola-derived known endoglucanases, and the present invention was completed.

The present invention relates to:

-   (1) a cellulase preparation comprising -   an endoglucanase derived from Zygomycetes, a protein in which a     cellulose binding domain is deleted in the endoglucanase, or a     modified or homologous protein thereof; and a reducing agent; -   (2) the cellulase preparation described in (1), wherein the     Zygomycetes is a microorganism belonging to genus Rhizopus, Mucor,     or Phycomyces; -   (3) a cellulase preparation comprising at least one of: -   (a) a protein consisting of any one of the amino acid sequences of     SEQ ID NOS: 1 to 6; -   (b) a protein consisting of an amino acid sequence in which a     cellulose binding domain is deleted in any one of the amino acid     sequences of SEQ ID NOS: 1 to 6; or -   (c) a protein consisting of an amino acid sequence in which one or     plural amino acids are deleted, substituted, inserted, or added, in     any one of the amino acid sequences of SEQ ID NOS: 1 to 6 or in an     amino acid sequence in which a cellulose binding domain is deleted     therein, and exhibiting an endoglucanase activity; and -   a reducing agent; -   (4) a cellulase preparation comprising a protein encoded by at least     one of: -   (a) a polynucleotide which encodes any one of the amino acid     sequences of SEQ ID NOS: 1 to 6; -   (b) a polynucleotide which encodes a protein consisting of an amino     acid sequence in which a cellulose binding domain is deleted in any     one of the amino acid sequences of SEQ ID NOS: 1 to 6; or -   (c) a polynucleotide which encodes a protein exhibiting an     endoglucanase activity and is complementary to a polynucleotide     which hybridizes under stringent conditions to a polynucleotide     which encodes a protein consisting of any one of the amino acid     sequences of SEQ ID NOS: 1 to 6 or a protein consisting of an amino     acid sequence in which a cellulose binding domain is deleted     therein; and -   a reducing agent; -   (5) the cellulase preparation described in any one of (1) to (4),     containing 0.01 to 50% by weight of the reducing agent in the     cellulase preparation; -   (6) the cellulase preparation described in any one of (1) to (5), in     which the reducing agent is sodium thiosulfate, sodium sulfite, or     thiourea; -   (7) the cellulase preparation described in any one of (1) to (6),     which is a granule not having a dustability or stabilized liquid; -   (8) a method of treating cellulose-containing fabric, comprising the     step of: -   treating the fabric with the cellulase preparation described in any     one of (1) to (7) to improve a property of the fabric; -   (9) the method described in (8), wherein the improvement of the     property of the fabric is a color clarification; -   (10) the method described in (8), wherein the improvement of the     property of the fabric is a removal of fuzz; -   (11) the method described in (8), wherein the improvement of the     property of the fabric is an addition of a stonewash-like appearance     and texture; -   (12) the method described in (8), wherein the improvement of the     property of the fabric is an improvement of touch and appearance; -   (13) the method described in (8), wherein the improvement of the     property of the fabric is a softening of the fabric; -   (14) the method described in any one of (8) to (13), wherein the     treatment of the fabric with the cellulase preparation is carried     out by soaking or rinsing the fabric; -   (15) a method of deinking waste paper, comprising the step of: -   treating the waste paper with the cellulase preparation described in     any one of (1) to (7) together with a deinking agent; and -   (16) a method of improving a freeness of paper pulp, comprising the     step of: -   treating the paper pulp with the cellulose preparation described in     any one of (1) to (7).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail hereinafter.

[1] Cellulase Preparation

The cellulase preparation of the present invention comprises one or more reducing agents and at least one of:

-   (1a) an endoglucanase derived from Zygomycetes; -   (1b) a protein in which a cellulose binding domain is deleted in the     Zygomycetes-derived endoglucanase (1a) [hereinafter sometimes     referred to as “CBD-deleted endoglucanase”]; -   (1c) a modified protein of the Zygomycetes-derived endoglucanase     (1a) or the CBD-deleted endoglucanase (1b) [hereinafter sometimes     simply referred to as “modified protein”]; or -   (1d) a homologous protein of the Zygomycetes-derived endoglucanase     (1a) or the CBD-deleted endoglucanase (1b) [hereinafter sometimes     simply referred to as “homologous protein”].

The term “endoglucanase” as used herein means endo-1,4-β-glucanase (EC 3.2.1.4), which has an activity of hydrolyzing the β-1,4-glucopyranosyl bond of β-1,4-glucan.

The term “endoglucanase activity” as used herein means a CMCase activity. The term “CMCase activity” as used herein means an activity of hydrolyzing carboxymethylcellulose (CMC; Tokyo Kasei Kogyo Co., Ltd.). When a solution containing a protein (enzyme) to be assayed and CMC is incubated for a predetermined period and the amount of reducing sugar released is measured, the amount of the enzyme producing the reducing sugar corresponding to 1 pmol of glucose per minute is defined as 1 unit of the CMCase activity.

The endoglucanase activity can be measured, for example, by the following procedure. That is, 0.5 mL of a solution containing a protein to be assayed is added to 0.5 mL of 2% CMC solution dissolved in 50 mmol/L acetate-sodium acetate buffer (pH 6.0), and the mixture is incubated at 50° C. for 30 minutes. A concentration of reducing sugar generated in the reaction mixture is measured by the 3,5-dinitrosalicylic acid method (DNS method). More particularly, after the incubation for 30 minutes, 3.0 mL of a DNS reagent is added to 1.0 mL of the reaction mixture, the whole is incubated in a boiling water bath for 5 minutes and diluted with 8.0 mL of distilled water, and the absorbance at 540 nm is measured. A calibration curve is drawn using glucose solutions prepared by stepwise dilution, and an amount of reducing sugar generated in the enzyme reaction mixture is determined as that of converted glucose. The activity is calculated by defining the amount of the enzyme producing the reducing sugar corresponding to 1 μmol of glucose per minute, as 1 unit.

The DNS reagent can be prepared in accordance with the disclosures in references such as Sakuzo Hukui, “Seikagaku Jikken-hou 1, Kangen-Tou no Teiryo-hou (Laboratory Manual for Biological Chemistry, Vol. 1, Assay of Reducing Sugar)”, pp. 19-20, Japan Scientific Societies Press, or by the following procedure. To 300 mL of 4.5% aqueous solution of sodium hydrate, 880 mL of 1% 3,5-dinitrosalicylic acid solution and 255 g of Rochelle salt are added (Solution A). To 22 mL of 10% aqueous solution of sodium hydrate, 10 g of crystalline phenol is added, and then water is added so as to dissolve it and adjust the volume to 100 mL (Solution B). Then, 6.9 g of sodium hydrogencarbonate is dissolved in 69 mL of Solution B, and Solution A is poured thereinto. The whole is mixed with stirring so as to dissolve the Rochelle salt, allowed to stand for 2 days, and then filtrated.

As the endoglucanase derived from Zygomycetes which may be used in the present invention, there may be mentioned, for example, endoglucanases derived from Rhizopus sp., Phycomyces sp. or Mucor sp. More particularly, for example, RCE I (SEQ ID NO: 1), RCE II (SEQ ID NO: 2), RCE III (SEQ ID NO: 3), MCE I (SEQ ID NO: 4), MCE II (SEQ ID NO: 5), or PCE I (SEQ ID NO: 6) disclosed in WO00/24879 may be used. As the CBD-deleted endoglucanase, proteins in which the cellulose binding domain is deleted in the above-mentioned endoglucanases, such as CBD-deleted endoglucanases disclosed in WO02/42474, may be used. The CBD-deleted endoglucanases lack the cellulose binding domain, and exhibit the endoglucanase activity.

Rhizopus oryzae CP96001, from which the above-mentioned RCE I, RCE II, and RCE III are derived, was domestically deposited in the International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (Address: AIST Tsukuba Central 6, 1-1, Higashi 1-chome Tukuba-shi, Ibaraki-ken 305-8566 Japan) on Apr. 21, 1997, and was transferred to an international deposit on Sep. 24, 1999. The international deposit number (a number in parenthesis [ ] following the international deposit number is a domestic deposit number) is FERM BP-6889 [FERM P-16201].

Mucor circinelloides CP99001, from which the above-mentioned MCE I and MCE II are derived, was domestically deposited in the International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (Address: AIST Tsukuba Central 6, 1-1, Higashi 1-chome Tukuba-shi, Ibaraki-ken 305-8566 Japan) on Jul. 2, 1999, and was transferred to an international deposit on Sep. 24, 1999. The international deposit number (a number in parenthesis [ ] following the international deposit number is a domestic deposit number) is FERM BP-6890 [FERM P-17446].

Phycomyces nitens CP99002, from which the above-mentioned PCE I is derived, was domestically deposited in the International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (Address: AIST Tsukuba Central 6, 1-1, Higashi 1-chome Tukuba-shi, Ibaraki-ken 305-8566 Japan) on Jul. 2, 1999, and was transferred to an international deposit on Sep. 24, 1999. The international deposit number (a number in parenthesis [ ] following the international deposit number is a domestic deposit number) is FERM BP-6891[FERM P-17447].

The proteins which can be used in the cellulase preparation of the present invention include not only RCE I, RCE II, RCE III, MCE I, MCE II, and PCE I, and the CBD-deleted endoglucanases as described above, but also modified and/or homologous proteins thereof.

The term “modified protein” as used herein means a protein exhibiting the endoglucanase activity and consisting of an amino acid sequence in which one or plural (for example, one to several tens, more particularly, 1 to 50, preferably 1 to 30, more preferably 1 to 9) amino acids are deleted, substituted, inserted, or added in the amino acid sequence of RCE I, RCE II, RCE III, MCE I, MCE II, or PCE I, or any one of the CBD-deleted endoglucanases thereof.

The term “homologous protein” as used herein means a protein exhibiting the endoglucanase activity and having an amino acid sequence encoded by a polynucleotide (base sequence) complementary to a polynucleotide (base sequence) which hybridizes under stringent conditions to a polynucleotide (base sequence) which encodes the amino acid sequence of RCE I, RCE II, RCE III, MCE I, MCE II, or PCE I, or any one of the CBD-deleted endoglucanases thereof. The term “polynucleotide” as used herein includes DNA and RNA, and DNA is preferable.

The term “stringent conditions” as used herein means conditions in which a probe comprising a base sequence which encodes a partial or full-length sequence of

-   (a) the amino acid sequence of RCE I, RCE II, RCE III, MCE I, MCE     II, or PCE I; -   (b) the amino acid sequence of any one of the CBD-deleted     endoglucanases thereof; or -   (c) any one of the modified proteins thereof hybridizes to a     polynucleotide which encodes a homologous protein, and the probe     does not hybridize to the endoglucanase NCE 4 gene (SEQ ID NO: 7)     described in WO98/03640 and the endoglucanase SCE 3 gene (SEQ ID     NO: 8) described in WO98/54322. In this connection, it should be     noted that the amount of each gene or polynucleotide used herein is     equivalent to the amount of each of the NCE 4 gene, the SCE 3 gene,     and the polynucleotide encoding a homologous protein.

More particularly, it means conditions in which, for example, using as a probe a labeled full-length DNA sequence encoding the amino acid sequence of RCE I, pre-hybridization is carried out at 42° C. for 1 hour according to the protocol attached to the ECL direct DNA/RNA labeling and detection system (Amersham), then the above probe is added thereto followed by hybridization at 42° C. for 15 hours, and thereafter, the resultant product is washed twice with 0.5×SSC (1×SSC; 15 mmol/L trisodium citrate, 150 mmol/L sodium chloride) containing 0.4% SDS and 6 mol/L urea at 42° C. for 20 minutes, and finally followed by washing the product twice with 5×SSC at room temperature for 10 minutes.

The above-mentioned “polynucleotide (base sequence) which encodes the amino acid sequence of RCE I, RCE II, RCE III, MCE I, MCE II, or PCE I” includes a polynucleotide in which codon usage and/or an intron recognition sequence are optimized in accordance with the type of a host cell used for transformation, such as the codon-optimized endoglucanase RCE I gene (SEQ ID NO: 9) described in WO00/24879.

As the modified or homologous protein, there may be mentioned, for example, a protein having an amino acid sequence having preferably an 80% or more homology, more preferably a 90% or more homology, still further preferably a 95% or more homology, most preferably a 98% or more homology, with that of RCE I, RCE II, RCE III, MCE I, MCE II, or PCE I, or any one of the CBD-deleted endoglucanases thereof. In this connection, the above values of homology may be values calculated using a known program for homology search, preferably values calculated using FASTA3 [Science, 227, 1435-1441 (1985); Proc. Natl. Acad. Sci. USA, 85, 2444-2448 (1988); http://www.ddbj.nig.ac.jp/E-mail/homology-j.html] in accordance with default parameters.

The “reducing agent”, which is contained in the cellulase preparation of the present invention, means a substance having an activity of reducing a molecule by accepting electrons from the molecule, thereby being itself oxidized. It is known that such reducing agents exhibit an activity of reducing and removing the remaining chlorine or the like in tap water. As the reducing agent used in the present invention, an inorganic reducing agent is preferable, and a substance which inhibits the enzyme activity cannot be used. Examples thereof include sulfurous acid, disulfurous acid, and thiosulfuric acid, and salts thereof, and thiourea. The reducing agents can be used alone or in a combination thereof.

The cellulose preparation of the present invention may comprise components which are conventionally contained in cellulase preparations such as excipients and/or preservatives. The form of the cellulose preparation may be solid or liquid. Examples of the form include powder, particulate, granule, non-dusting granule and liquid formulation.

The non-dusting granule (preferably a granule not having a dustability) that is one form of cellulase preparation can be produced according to the common dry granulation method. That is to say, powder cellulase enzyme is mixed with one or plural substances selected from the group comprising inorganic salts such as sodium sulfate or sodium chloride which are neutral and do not have an effect on the endoglucanase activity; minerals such as bentonite or montmorillonite which do not have an effect on the endoglucanase activity; neutral organic substances such as starch or powder cellulose; and surfactants. Thereafter, the powders or the finely suspended suspension of one or plural reducing agents which improve the effects of endoglucanase are added to the mixture, and then the obtained product is fully mixed or kneaded.

Depending on the situation, a synthetic polymer such as polyethylene glycol or a natural polymer such as starch, which binds solids, is optionally added to the mixture and further kneaded. Thereafter, granulation is carried out by extrusion molding, using, for example, a disk pelleter, and the obtained molded material is then converted into a spherical form using a marumerizer followed by drying, so that non-dusting granules can be produced. Naturally, it is also possible to coat the surface of granules with a polymer or the like to control the permeation of oxygen or water. In this case, one or plural reducing agents which improve the effect of endoglucanase can be added to the cellulase preparation at a ratio of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, more preferably 0.1 to 10% by weight.

Further, the liquid preparation (preferably stabilized liquid) can be prepared by blending an endoglucanase stabilizer such as a synthetic or natural polymer with a cellulase enzyme solution and, if necessary, adding inorganic salts and/or a synthetic preservative. In this case, one or plural reducing agents which improve the effect of endoglucanase can be added. Similar to the case of the non-dusting granule, one or plural reducing agents which improve the effect of endoglucanase can be added to the cellulase preparation at a ratio of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, more preferably 0.1 to 10% by weight.

[2] Method of Treating Fabric

The method of treating fabric according to the present invention comprising the step of: treating cellulose-containing fabric with the above-mentioned cellulase preparation.

The following properties of cellulose-containing fabric can be improved by the present treatment method:

-   (1) Color clarification of colored cellulose-containing fabric; -   (2) Removal of fuzz (reduction of the rate of the formation of fuzz,     and reduction of fuzz); -   (3) Providing of localized color variation to colored     cellulose-containing fabric, that is, providing a stonewash-like     appearance and texture to colored cellulose-containing fabric,     typically jeans; -   (4) Improvement of the touch and appearance of fabric by reducing     weight; and -   (5) Softening of fabric (reduction of stiffness).

More particularly, the method of treating fabric according to the present invention can be carried out by adding the cellulase preparation of the present invention into water in which fabric is or will be soaked, for example, during a soaking or rinsing of fabric.

Conditions such as contact temperature or the amount of endoglucanase may be appropriately determined in accordance with various other conditions. For example, when reducing the rate of the formation of fuzz or reducing fuzz of the cellulose-containing fabric, the fabric can be treated at a temperature of approximately 30 to 60° C., using 0.2 μg/mL or more of reducing agents and endoglucanases in a protein concentration of 0.001 to 20 mg/L. One or more reducing agents may be added, taking into consideration economical effects, so long as an amount thereof is 0.2 μg/mL or more and the reducing agent does not inhibit the enzyme activity. Preferably 0.2 to 500 μg/mL, more preferably 0.3 to 150 μg/mL thereof may be used.

When providing a localized color variation to colored cellulose-containing fabric, the fabric can be treated at a temperature of approximately 40 to 60° C., using 0.2 μg/mL or more of reducing agents and endoglucanases in a protein concentration of 0.01 to 100 mg/L. One or more reducing agents may be added, taking into consideration economical effects, so long as an amount thereof is 0.2 μg/mL or more and the reducing agent does not inhibit the enzyme activity. Preferably 0.2 to 500 μg/mL, more preferably 0.3 to 150 μg/mL thereof may be used.

In a processing of reducing weight to improve the touch and appearance of the cellulose-containing fabric, the fabric can be treated at a temperature of approximately 30 to 60° C., using 0.2 μg/mL or more of reducing agents and endoglucanases in a protein concentration of 0.001 to 100 mg/L. One or more reducing agents may be added, taking into consideration economical effects, so long as an amount thereof is 0.2 μg/mL or more and the reducing agent does not inhibit the enzyme activity. Preferably 0.2 to 500 μg/mL, more preferably 0.3 to 150 μg/mL thereof may be used.

The protein concentration of each type of endoglucanase can be calculated, for example, by HPLC analysis using TSKgel TMS-250 column (4.6 mm I.D. ×7.5 cm) (TOSOH Corporation). The HPLC analysis involves loading acetonitrile in 0.05% TFA (trifluoroacetic acid) with a linear concentration gradient of 0% to 80% at a flow rate of 1.0 mL/min so as to elute each type of endoglucanase, and calculating the protein concentration from the peak area at UV 280 nm. For example, a purified NCE4, the protein concentration of which is previously determined by a Protein Assay Kit (BioRad Laboratories), is subjected to the HPLC analysis in the same manner as above, so that it can be used as a standard. The purified NCE4 can be obtained, for example, by cultivating Humicola insolens MN200-1 and purifying it from the culture, in accordance with the method described in WO98/03640. As a standard for the determination of a protein concentration in the Protein Assay Kit, for example, Albumin Standard (Bovin serum albumin, fraction V; PIERCE) can be used.

-   [3] Method of Deinking Waste Paper

The method of deinking waste paper according to the present invention comprises the step of:

-   treating the waste paper with the above-mentioned cellulase     preparation together with a deinking agent.

More particularly, the present method can be carried out by treating waste paper with the cellulase preparation of the present invention together with a deinking agent, in a deinking step in a process of producing recycled paper from waste paper. The present method enables the deinking of waste paper, and thus the whiteness of waste paper can be improved. Waste paper which can be treated by the present method includes all types of common waste paper, for example, used newspaper, used magazine paper, and low to middle grade printed used paper which comprise mechanical pulp and chemical pulp; used wood-free paper comprising chemical pulp; and printed waste paper thereof such as coating paper. The deinking agent means an agent commonly used in the deinking of waste paper. Examples of the deinking agent include sodium chloride, alkalis such as sodium carbonate, sodium silicate, hydrogen peroxide, phosphates, anionic or nonionic surfactants, scavengers such as oleic acid, and assistant agents such as a pH stabilizer, a chelating agent, or a dispersing agent.

[4] Method of Improving Freeness of Paper Pulp

The method of improving the freeness of paper pulp according to the present invention comprises the step of:

-   treating the paper pulp with the above-mentioned cellulase.     preparation.

More particularly, the present method can be carried out by treating paper pulp with the cellulase preparation of the present invention. Examples of paper pulp which can be treated by the present method include waste paper pulp, recycled paperboard pulp, kraft pulp, sulfite pulp, thermo-mechanical treatment pulp, and other high-yield pulp.

EXAMPLES

The present invention now will be further illustrated by, but is by no means limited to, the following Examples.

All publications and patent applications mentioned in the present specification are herein incorporated by reference.

Example 1 Comparison Among Improvement Ratios of Fuzz-Removing Activities of Various Types of Cellulases by Addition of Reducing Agent

The cultivation of Rhizopus oryzae, Mucor circinelloides, and Phycomyces nitens, and the purification of RCE I, MCE I, and PCE I endoglucanases from the cultures were carried out by the method described in WO00/24879.

The cultivation of Humicola insolens MN200-1 and the purification of NCE4 endoglucanase from the culture were carried out by the method described in WO98/03640. The strain was domestically deposited in the International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (Address: AIST Tsukuba Central 6, 1-1, Higashi 1-chome Tukuba-shi, Ibaraki-ken 305-8566 Japan) on Jul. 15, 1996, and was transferred to an international deposit on Jun. 13, 1997. The international deposit number (a number in parenthesis [ ] following the international deposit number is a domestic deposit number) is FERM BP-5977 [FERM P-15736].

The cultivation of Trichoderma viride MC300-1 was carried out by the method described in WO98/54332. The strain was domestically deposited in the International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (Address: AIST Tsukuba Central 6, 1-1, Higashi 1-chome Tukuba-shi, Ibaraki-ken 305-8566 Japan) on Sep. 9, 1996, and was transferred to an international deposit on Aug. 11, 1997. The international deposit number (a number in parenthesis [ ] following the international deposit number is a domestic deposit number) is FERM BP-6047 [FERM P-15842].

Fuzz-removing treatment of a cotton knit fabric with fuzz formed in a large washer (a fabric of 6 cm×8 cm from Cotton Smooth Knit No. 3900, Nitto Boseki Co., Ltd. dyed brown by reactive dyeing in Tsuyatomo-Senko) was carried out using each of the obtained culture supernatants under the following conditions:

(Test Conditions)

-   -   Testing machine: Launder Meter L-20 (Daiei Kagaku Seiki MFG.,         Japan)     -   Temperature: 58° C. (only the Trichoderma viride culture         supernatant); 40° C. (all other enzyme solutions)     -   Time: 120 minutes     -   Amount of reaction solution: 100 mL     -   Reaction pH: pH 4.5 (5 mmol/L acetate buffer) (only the         Trichoderma viride culture supernatant pH 7.0 (5 mmol/L         phosphate buffer) (all other enzyme solutions). All the buffers         were prepared using tap water.     -   Type and amount of reducing agent: 1.2 μg/mL sodium thiosulfate         pentahydrate (Wako Pure Chemical Industries, Co., Ltd.)

To each of the treating solutions, four of about 16 g rubber balls were added together with each enzyme solution.

The amount of the enzyme solution required to remove approximately 50% of the formed fuzz on the basis of visual evaluation was determined in each of both cases of adding and not adding the reducing agent. Thereafter, a value was obtained by dividing the amount of the enzyme solution required to remove approximately 50% of the fuzz when not adding the reducing agent by the amount when adding the reducing agent, and the obtained value was defined as an improvement ratio of the fuzz-removing activity by the addition of the reducing agent. The results are shown in Table 1. TABLE 1 Improvement ratio of fuzz- removing activity by addition Enzyme solutions of reducing agent (fold) Humicola insolens culture 1.2 supernatant Trichoderma viride culture 1.1 supernatant Purified NCE4 1.2 Purified RCEI 5.0 Purified MCEI 3.5 Purified PCEI 2.5

From the results of Table 1, it is found that the fuzz-removing activity of RCE I, MCE I, and PCE I, which are endoglucanases derived from Zygomycetes, is improved by the addition of the reducing agent at a level far higher than culture supernatants (i.e., cellulase) derived from Humicola insolens and Trichoderma viride.

Example 2 Improvement Effect of Fuzz-Removing Activity of RCE I Expressed in Humicola by Addition of Various Reducing Agents

RCE I endoglucanase was expressed in Humicola insolens in accordance with the method described in Examples D3 and D4 of WO00/24879. Fuzz-removing treatment of a cotton knit fabric with fuzz formed in a large washer (a fabric of 6 cm 8 cm from Cotton Smooth Knit No. 3900, Nitto Boseki Co., Ltd. dyed brown by reactive dyeing in Tsuyatomo-Senko) was carried out using the obtained culture supernatant under the following conditions:

(Test Conditions) Testing machine: Launder Meter L-20 (Daiei Kagaku Seiki MFG., Japan) Temperature: 40° C. Time: 120 minutes Amount of reaction solution: 100 mL Reaction pH: pH 7.0 (5 mmol/L phosphate buffer; prepared using tap water) Amount of reducing agent: 1.2 μg/mL Type of reducing agent: sodium thiosulfate pentahydrate (Wako Pure Chemical Industries, Co., Ltd.); sodium sulfite (anhydride) (Wako Pure Chemical Industries, Co., Ltd.); and thiourea (Kanto Kagaku, Co., Ltd.).

To each of the treating solutions, four of about 16 g rubber balls were added together with the enzyme solution.

The amount of the enzyme solution required to remove approximately 50% of the formed fuzz on the basis of visual evaluation was determined in each of both cases of adding and not adding the various reducing agents. Thereafter, a value was obtained by dividing the amount of the enzyme solution required to remove approximately 50% of the fuzz when not adding the reducing agent by the amount when adding each of the various reducing agents, and the obtained value was defined as an improvement ratio of the fuzz-removing activity by the addition of each of the various reducing agents. The results are shown in Table 2. TABLE 2 Improvement ratio of fuzz- removing activity by addition of Reducing agents reducing agent (fold) Sodium thiosulfate 5.0 pentahydrate Sodium sulfite 5.0 (anhydride) Thiourea 5.0

From the results of Table 2, it is found that the fuzz-removing activity of the culture supernatant obtained by expressing and secreting RCE I in Humicola insolens was improved by any of the above reducing agents.

Example 3 Improvement Effect of Fuzz-Removing Activity of RCE I Expressed in Humicola by addition of Reducing Agent with Various Concentrations

-   RCE I endoglucanase was expressed in Humicola insolens in accordance     with the method described in Examples D3 and D4 of WO00/24879.     Fuzz-removing treatment of a cotton knit fabric with fuzz formed in     a large washer (a fabric of 6 cm×8 cm from Cotton Smooth Knit No.     3900, Nitto Boseki Co., Ltd. dyed brown by reactive dyeing in     Tsuyatomo-Senko) was carried out using the obtained culture     supernatant under the following conditions:

(Test Conditions) Testing machine: Launder Meter L-20 (Daiei Kagaku Seiki MFG., Japan) Temperature: 40° C. Time: 120 minutes Amount of reaction solution: 100 mL Reaction pH: pH 7.0 (5 mmol/L phosphate buffer; prepared using tap water) Type of reducing agent: sodium thiosulfate pentahydrate (Wako Pure Chemical Industries, Co., Ltd.) Amount of reducing agent: 0.15 to 150 μg/mL

To each of the treating solutions, four of about 16 g rubber balls were added together with the enzyme solution.

The amount of the enzyme solution required to remove approximately 50% of the formed fuzz on the basis of visual evaluation was determined in each of cases of adding various concentrations of the reducing agent. Thereafter, a value was obtained by dividing the amount of the enzyme solution required to remove approximately 50% of the fuzz when not adding the reducing agent by the amount when adding each of various concentrations of the reducing agent, and the obtained value was defined as an improvement ratio of the fuzz-removing activity by the addition of each of various concentrations of the reducing agent. The results are shown in Table 3. TABLE 3 Improvement ratio of fuzz-removing Amount of reducing activity by addition of reducing agent (μg/ml) agent (fold) 0.15 1.0 0.3 2.5 0.6 4.5 0.9 5.0 1.2 5.0 1.8 4.5 3.0 4.5 6.0 4.5 15.0 4.5 30.0 4.5 60.0 4.0 150.0 4.0

From the results of Table 3, it is found that the fuzz-removing activity of the culture supernatant obtained (21) by expressing and secreting RCE I in Humicola insolens was improved by the addition of the reducing agent having a wide range of concentration from 0.3 μg/mL to 150 μg/mL or more.

Example 4: Production of RCE I Cellulase Preparation Comprising Reducing Agent

After mixing the following raw materials by the mixing ratios as described in Table 4, an appropriate amount of water was added thereto, and the mixture was kneaded. The obtained product was subjected to a disk pelleter for molding, and the product obtained by injection molding was converted in a particle form using a marumerizer (Fuji Paudal Co., Ltd.) followed by drying and sieving the product so as to obtain a granulated product. TABLE 4 Mixing ratio Raw materials (%) Sodium thiosulfate 1 S-220 (nonionic surfactant manufactured by 10 NOF Corporation) RCE I cellulase powder product 5 Magnesium chloride 0.5 Monopotassium phosphate (Wako Pure Chemical 2 Industries, Co., Ltd.) Dipotassium phosphate (Wako Pure Chemical 1 Industries, Co., Ltd.) Corn starch (Shikishima Starch Co.) 80.5

The RCE I cellulase powder product was prepared by concentrating the culture supernatant of RCE I expressed in Humicola insolens using ultrafiltration, according to the method described in Examples D3 and D4 of WO00/24879, followed by spray drying.

INDUSTRIAL APPLICABILITY

The present invention provides a cellulase preparation having a dramatically improved Zygomycetes-derived endoglucanase activity by adding a reducing agent into the preparation. When the cellulase preparation is used in the treatment of fabric (such as the reduction of fuzz of cellulose-containing fabric, the improvement of touch and appearance, the color clarification, localized color variation, or softening), the deinking of waste paper, or the processing of improving the freeness of paper pulp, each of the above treatments can be carried out with a less amount of enzyme, thereby significantly reducing cost.

Free Text in Sequence Listing

Features of “Artificial Sequence” are described in the numeric identifier <223>in the Sequence Listing. More particularly, the base sequence of SEQ ID NO: 9 is a codon-optimized sequence corresponding to RCE I protein (SEQ ID NO: 1).

Although the present invention has been described with reference to specific embodiments, various changes and modifications obvious to those skilled in the art are possible without departing from the scope of the appended claims. 

1. A cellulase preparation comprising an endoglucanase derived from Zygomycetes, a protein in which a cellulose binding domain is deleted in the endoglucanase, or a modified or homologous protein thereof; and a reducing agent.
 2. The cellulase preparation according to claim 1, wherein the Zygomycetes is a microorganism belonging to genus Rhizopus, Mucor, or Phycomyces.
 3. A cellulase preparation comprising at least one of: (a) a protein consisting of any one of the amino acid sequences of SEQ ID NOS: 1 to 6; (b) a protein consisting of an amino acid sequence in which a cellulose binding domain is deleted in any one of the amino acid sequences of SEQ ID NOS: 1 to 6; or (c) a protein consisting of an amino acid sequence in which one or plural amino acids are deleted, substituted, inserted, or added, in any one of the amino acid sequences of SEQ ID NOS: 1 to 6 or in an amino acid sequence in which a cellulose binding domain is deleted therein, and exhibiting an endoglucanase activity; and a reducing agent.
 4. A cellulase preparation comprising a protein encoded by at least one of: (a) a polynucleotide which encodes any one of the amino acid sequences of SEQ ID NOS: 1 to 6; (b) a polynucleotide which encodes a protein consisting of an amino acid sequence in which a cellulose binding domain is deleted in any one of the amino acid sequences of SEQ ID NOS: 1 to 6; or (c) a polynucleotide which encodes a protein exhibiting an endoglucanase activity and is complementary to a polynucleotide which hybridizes under stringent conditions to a polynucleotide which encodes a protein consisting of any one of the amino acid sequences of SEQ ID NOS: 1 to 6 or a protein consisting of an amino acid sequence in which a cellulose binding domain is deleted therein; and a reducing agent.
 5. The cellulase preparation according to claim 1, containing 0.01 to 50% by weight of the reducing agent in the cellulase preparation.
 6. The cellulase preparation according to claim 1, in which the reducing agent is sodium thiosulfate, sodium sulfite, or thiourea.
 7. The cellulase preparation according to claim 1, which is a granule not having a dustability or stabilized liquid.
 8. A method of treating cellulose-containing fabric, comprising the step of: treating the fabric with the cellulase preparation according to claim 1 to improve a property of the fabric.
 9. The method according to claim 8, wherein the improvement of the property of the fabric is a color clarification.
 10. The method according to claim 8, wherein the improvement of the property of the fabric is a removal of fuzz.
 11. The method according to claim 8, wherein the improvement of the property of the fabric is an addition of a stonewash-like appearance and texture.
 12. The method according to claim 8, wherein the improvement of the property of the fabric is an improvement of touch and appearance.
 13. The method according to claim 8, wherein the improvement of the property of the fabric is a softening of the fabric.
 14. The method according to claim 8, wherein the treatment of the fabric with the cellulase preparation is carried out by soaking or rinsing the fabric.
 15. A method of deinking waste paper, comprising the step of: treating the waste paper with the cellulase preparation according to claim 1 together with a deinking agent.
 16. A method of improving a freeness of paper pulp, comprising the step of: treating the paper pulp with the cellulase preparation according to claim
 1. 